For millennia, various types of cells have been known to be useful to manufacture products. Among these cells are bacteria, yeast, plant, insect and mammalian cells. For example, beer and wine were made with the use of yeast cells by the ancient Egyptians. Many of the useful cells have robust physiology and structure and can be grown successfully as suspension cultures in stationary vats. If it is necessary to mix the culture in order to provide air or nutrients, paddles and/or air lifts can be used. However, these methods of mixing and aeration cause shearing and destruction of more delicate cells such as insect and mammalian cells, and bacterial, plant and yeast protoplasts. Such suspension cultures are not useful for cells that require attachment to a substrate for growth.
Roller bottles, in which the culture is mixed by the rotation of culture bottles on a roller, have long been used to bathe cells gently in nutrient and to expose them to air. Roller bottles also provide a surface for cells that require a substrate on which to attach.
However, a serious drawback to roller bottles is that in order to add fresh culture medium or to withdraw spent medium, it is necessary to open the bottles or to add/withdraw fluid by syringe, time-consuming and labor intensive tasks, which increase the possibility of contamination.
Roller bottle cell culture is well known. A roller table of any desirable size is equipped with a series of rollers. When these rollers are driven by a motor to rotate, bottles placed within the declivity between two rollers will rotate, providing gentle mixing of the bottle contents. Mixing is improved in some models by molding baffles on the inside surface of the bottle or providing paddles.
Comparison of monascus pigments produced by the fungus Monascus anka show the advantages of mixed versus stationary cultures. Cells were grown in batch-submerged, agar surface and roller bottle culture. The cells grown in the roller bottle produced a pigment yield about ten times greater than that of the other two systems. The time required for maximum pigment production was four days in the roller bottle compared to seven days for the batch-submerged and agar cultures.
The addition of nutrient also improves product yield. With hybridomas grown in a typical batch culture, the number of viable cells varies with time. A batch culture begins with a lag phase, where there is no increase in cell number. Following the lag phase, a period of exponential growth occurs in the logarithmic phase. A stationary phase follows where the cell population is at a maximum size. Finally, a decline in cell number occurs in the death phase. Reuveny et al investigated monoclonal antibody production in four different systems using a single hybridoma cell line. The four systems employed were fed-batch, semi-continuous, two-stage semi-continuous and perfusion. Each was compared to batch culture.
In the fed-batch system, a spinner flask was initially inoculated with 60 milliliters of culture media. When the cell density reached a value just below its known peak in batch mode, daily addition of six milliliters of fresh media was added to the culture. This method maintained the viable cell concentration between five and eight times the level found in batch cultures. The amount of antibody present on day eight was almost double that found in the batch system while the total media used was approximately equal. This fed-batch method of propagation resulted in an average antibody production rate of 27 micrograms per milliliter per day, as compared to a daily average production rate of 15 micrograms per milliliter per day in the batch control.
The semi-continuous system employed a system in which 100 milliliter cultures in spinner flasks were fed twice daily. Each day prior to the first feeding, a volume equivalent to the amount of media to be fed that day was removed. Fresh media was then added every 12 hours. Four tests were run where media volumes ranging from five percent to 40 percent of the total culture volume was replaced daily. The replacement rate of 20 percent resulted in the highest average daily antibody production rate equal to 34 milligrams per milliliter per day. The two-stage semi-continuous system involved a semi-continuous feeding strategy as above, but, instead of harvesting antibody from the cultures that were removed daily, a second stage vessel was used to feed the removed culture in order to maintain cell viability. With this second stage, a productivity rate of 62 micrograms per milliliter per day was obtained.
The perfusion culture system was constructed by mounting a cylindrical filter with five micrometer openings around the stirring shaft of a one liter reactor. Since the hybridomas are larger than ten micrometers, the screen retains the cells and allows cell-free supernatant to collect in the inner part of the rotating cylinder. Continuous perfusion occurred by controlled addition of fresh media to the culture on the outside of the cylinder while pumping out cell-free supernatant from within the cylinder at the same rate. Perfusion was initiated when cells reached their maximum density in batch. The feeding was initiated at a rate of 27 percent of total reactor volume per day. The rate had to be increased daily until the cell density leveled off at a maximum value. The feeding rate reached 1790 percent of the reactor volume per day (1,700 milliliters per day in the one liter reactor). Antibody production was high, averaging 660 micrograms per milliliter per day.
Many other attempts have succeeded in devising systems that maintain viability and production. In general, these systems have either been complex and expensive in initial apparatus cost and or simple but very labor-intensive.
The need remains to attain a high level of cell growth and/or production, simply, without cumbersome and expensive equipment or high labor input.
This invention provides a reservoir chamber having a spiroidal pump formed of tubing placed in a spiral on the inside surface of the reservoir chamber. The inlet end of the tubing is open to the reservoir while the outlet end is open to a growth chamber. The reservoir chamber is attached to the growth chamber so that the two chambers rotate synchronously on their horizontal axes when placed on a moving roller table. The reservoir chamber and growth chamber may be formed by dividing a roller bottle with an partition that is impermeable to fluids. Alternatively, the reservoir chamber may be a separate bottle attached to the growth chamber. Each bottle is fitted with a removable cap, which is preferably a filter of such a mesh as to allow the passage of fluids but not particles such as bacteria. The growth chamber is partially filled with inoculated culture medium, while the reservoir chamber is partially filled with replacement medium. Upon rotation, the inlet end of the tubing will fill first with replacement medium and then with air as the chamber is rotated. The xe2x80x9cplugsxe2x80x9d of air and medium move along the tubing with continued rotation and empty into the growth chamber. The volumetric feeding rate at which fresh replacement medium is moved from the reservoir chamber to the roller bottle can be varied by varying the diameter of the tubing and/or the rotation rate.
Also provided is a collection bottle, attached to the growth chamber distal to the attachment of the reservoir chamber, so that when the culture medium in the growth chamber reaches a predetermined level, the medium will overflow into the collection bottle. Preferably, the collection bottle is fitted with a valve and connect/disconnect fitting so that it can be removed and replaced with another collection bottle. If the reservoir chamber is a bottle separate from the growth chamber, it is advantageous to fit the reservoir chamber also with a valve and connect/disconnect fitting. Thus this invention provides a means of feeding and harvesting the contents of the system quickly and conveniently without opening the growth chamber.